The invention relates to a selection method and the products resulting from the method. More specifically the invention relates to a method for acquiring one or more binding structures against a target structure by means of a first library of one or more binding structures linked to genetic and/or other identifying information.
The phage display technology offers a powerful means to select anti-body fragments with rare specificities from large libraries (1). Antibody libraries from immune, naive and semisynthetic sources of 1010 diversity and beyond (2-4) can efficiently be examined by the antigen driven selection principle. Typically, for pure protein antigens or haptens, affinity chromatography or panning techniques have been used to achieve enrichment of one hundred to ten-thousand fold by each selection round (5-7). Recently, selection systems based on complex solid-phase antigens such as viable bacterial cells (8), human erythrocytes (9) or other cell suspensions (10-13) have been described. These introduce the use of antibody phage selection to xe2x80x9cdissectxe2x80x9d a cell surface by cloning antibodies specific to individual cell surface antigens. This can be either random or be directed to a phenotypically defined subset of antigens. One example of a situation where the method described could be highly advantageous is research with the aim to identify new target structures for targeted tumor therapy.
The target tumor phenotype for antibody-guided tumor immunotherapy is typically an invasive/metastatic tumor cell population. Such tumor cells could not be expected to be adequately represented by cultured tumor cell lines primarily selected for proliferation in an artificial culture environment Instead, continued in vitro culture would aggravate phenotypic drift and downregulation of expression of tumor-associated molecules that was sustained in vivo by growth architecture and by the influence of soluble or mesenchymal tissue constituents (15,16). Selection of antibodies for tumor targeting, being critically dependent on the homogenous and abundant expression of the target epitope should preferentially be performed using the authentic tumor cell phenotype during both the antigen driven selection stage and the screening of individual clones.
Thus, successful selection from large libraries of antibody phage specific to known purified antigens has been demonstrated by a number of laboratories. It has also been shown that intact bacterial or mammalian cells, which represent a more complex surface of antigens, can be used for antibody phage selection. However, the use of cell suspensions for phage selection can only generate reagents to cell surface antigens: other components of a tissue such as the extracellular matrix, inflammatory infiltrates, vasculature and components of angiogenesis and intracellular antigens can not be reached by the cell selection approach. In addition, culturing of cells through several passages will introduce selection and phenotypic changes compared to the original cell isolated from a specific tissue environment. Accordingly, there is a demand for a method which could be particularly useful for the identification of antigens which are only expressed in vivo. Such a method should overcome a major limitation of currently used selection protocols.
The purpose of the present invention is to provide an extension of the application of phage technology for the selection of antibodies to complex antigens, thus making it generally applicable to identify antibodies directed against a number of important and/or novel target antigens and epitopes which are not accessible in in vitro culture systems, which would facilitate identification and dissection of antigens which are exclusively expressed in vivo. Of course, the method could generate antibodies to any target structure displayed within the tissue section.
These include antigens whose expression is induced by epithelial cell-mesenchymal cell and cell-matrix interactions, or is tightly regulated spatially or temporally during embryonic development and by a specific patho-physiological process, e.g. tumor progression (14).
A further purpose of the invention is to provide a method with a broad applicability as an analytical tool in studies of cell and tissue development and of antigenic phenotypes associated with tissue pathology of various conditions. Hereby is provided a novel and unique means for a much less biased dissection of specificities among a broad range of tissue expressed antigens analogous to the use of naive antibody libraries for selection of reagents to single antigens (23).
Still a further purpose of the invention is to include in the method by means of negative selection a specific removal of undesired antibodies in order to promote the enrichment of phenotype specific specificities.
In order to achieve these purposes the method according to the invention has obtained the characterizing features of claim 1.
The method according to the invention should have a broad applicability in a variety of research fields (e.g. in the study of embryogenesis, angiogenesis and tumor biology) where novel antibody specificities could be used to define temporal or spatial expression of normal or pathologic tissue phenotypes. The phage selection method described below and applied to selection of melanoma reactive scFv antibodies on sections of metastatic melanoma tissue should thus only be considered and construed as a specific example of the present invention. Thus, the usefulness of the method should not be restricted to identification of tumor associated antigens, but rather be generally applicable for the in situ identification of target structure expression patterns in both normal and pathological tissues.